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Objective:To investigate the levels of C/EBPγ mRNA in liver cancer patients and the effect and mechanism of C/EBPγ on proliferation and migration of HepG2 cells.Methods:This experiment was performed in Department of Clinical Laboratory of Shanghai Songjiang District Central Hospital in vitro from October of 2018 to April of 2019. The pLKO.1 was the control group and shg1/shg2 were interference groups. The transcriptional levels of C/EBPγ in liver cancer were analyzed through Oncomine database and real-time PCR. HepG2 and Hep3B cells were cultivated in DMEM media with 10% FBS at 37℃ and 5% CO 2. Adenovirus vector shC/EBPγ-pLKO.1 was constructed and infected 293T cell combined with packaging carrier pMD2G, pMDLG/REE and pRSV/Rev. The empty vector pLKO.1 served as control. The virus supernatant was collected at 12 h and infected HepG2 cells four times. Puromycin were added at the concentration of 2 μg/mL and cells were selectively cultured for 48-72 h. The puromycin concentration was changed into 1 μg/mL after the selection. Growth curves were used to detect cell proliferation. ROS levels were determined by using DFH-DA assays. The oxidative stress related genes transcription changes were detected by using real-time PCR. The migration of HepG2 cells was assayed through scratch assay. NAC (1 mmol/L) were added to cells against oxidative stress. Results:The mRNA levels of C/EBPγ were much higher in liver cancer patients and cells than normal controls. The proliferation rates of C/EBPγ interference groups at 4 d (shg1: 1.93±0.70, shg2: 1.28±0.40) and 6 d (shg1: 3.05±0.90, shg2: 1.31±0.60) were lower than control groups (4 d: 6.18±0.80, 6 d: 22.41±2.56) significantly ( F=1 507.971, P<0.05) . Reactive oxygen species (ROS) levels were elevated in C/EBPγ interference groups and the transcription level of oxidative stress related genes (HPRT1, NQO1-tv2 and NQO1-tv4) was increased at 12 h and 24 h when the cells were cultured without serum. The scratch assays showed that non-healing area in C/EBPγ interference groups (shg1: 174 922.58±1 239 376.00,shg2: 1 374 656.00±248 882.79) was larger than control groups (66 690.82±1 278 954.00) ( F=39.871, P<0.05) . When the antioxidant NAP was added, the proliferation rates were elevated at 6 h and the ROS levels were decreased at 0.5 h, 1 h and 3 h ( F=7.587, 4.657, 3.903, P<0.05) . Conclusions:C/EBPγ mRNA levels were increased in liver cancer. When C/EBPγ was disturbed in HepG2 cells, the proliferation and migration were decreased, the ROS levels and oxidative stress related genes were elevated significantly, which indicated that C/EBPγ promoted proliferation and migration through reducing ROS levels in liver cancer.
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Objective@#To investigate the effects and mechanism of hepatitis B virus x protein (HBx) on human hepatocellular carcinoma cells proliferation.@*Methods@#Eukaryotic expression vector HBx-pEGFP-C1 was constructed. HepG2 cells were transfected transiently using Lipofectamine 2000. HBx expression in transfected cells were measured by RT-PCR and Western blot. Cells proliferation and apoptosis were detected by using growth curves and TUNEL staining. The protein levels of caspase-3, p-p38, p-Akt and p-JNK were measured by Western blot.@*Results@#HBx was successfully expressed in HepG2 cells. Growth curve result showed that HBx promoted cell proliferation (t=-0.8999, P=0.012). Compared with control group, the levels of p-p38(24 h) (t=- 11.058, P=0.0004) and p-JNK(48 h) (t=- 15.022, P=0.0001) in HBx-pEGFP-C1 group were increased significantly. There is no significant difference between the two groups′ apoptosis.@*Conclusions@#Transient overexpression of HBx promoted human hepatic carcinoma cells proliferation and activated the p38 and JNK signalling pathway.
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RNAs are known to regulate diverse biological processes, either as protein-encoding molecules or as non-coding RNAs.However, a new class of bi-functional RNAs, carrying both protein coding and RNA-intrinsic functions, have been identified and termed as 'CncRNAs'.This review discuss three major genomic sources of CncRNAs and describe the dual characteristics and functional mechanisms of them, providing a new perspective to further understand the complexity of the transcriptomics and genomes and the complex gene-regulatory network in organisms.
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Objective To evaluate the expression and clinical significance of miRNA in plasma of patients with primary biliary cirrhosis.Methods Plasma from 19 PBC patients,10 healthy volunteers and 10 viral hepatitis patients were selected from Shanghai Songjiang Hospital from december 2010 to January 2013.Among them 3 PBC patients' plasma and 3 healthy volunteers' plasma were detected by miRNA microarray for miRNA expression profile examination.Real-time PCR was used to verify the results of microarray,miRNA target gene predictior software was used to predict the target genes of differentially expressed miRNA.ROC was used to determine the clinical value of plasma miRNA.Results According to microarray,a total of 16 miRNAs were found to be differentially expressed.As revealed by qRT-PCR,the expression of miRNA-92a-3p and miRNA-4516 decreased while the expression of miRNA-572 and miRNA-575 were up-regulated in PBC group compared with healthy controls (P ＜ 0.05).In comparison with nonPBC cirrhosis group,only miRNA-92a-3p and miRNA-4516 were down-regulated (P ＜ 0.05).The area under the curve (AUC)of miRNA-92a-3p for the diagnosis and differential diagnosis of PBC were 0.92 and 0.84,respectively.while for The area under the ROC curve of miRNA-4516,the AUC for diagnosis and differential diagnosis PBC were 0.89 and 0.76,respectively.The optimal cut-off values for identifying PBC from healthy controls were defined as 1.26 ng/μl.for miRNA-92a-3p (sensitivity,92％ ;specificity,80％)and 1.16ng/ul for miRNA-4516 (sensitivity,85％ ;specificity,70％)respectively.The optimal cut-off values for identifying PBC from viral hepatitis were defined as 1.08 ng/μl.for miRNA-92a-3p (sensitivity,89％ ; specificity,81％)and 1.06 ng/μl for miRNA-4516 (sensitivity,77％ ;specificity,68％).Conclusion The results indicate that plasma from patients with PBC has a unique miRNA exprssion profile and these differentially expressed miRNA can be used as clinical biomarkers of PBC.
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Objective To understand the status of mycoplasma infection and drug susceptibility to provide reference basis for clinical rational drug use.Methods The reagents kits produced by Shanghai Aopu Biopharmaceutical Co.,Ltd.were adopted to per-form the mycoplasma culture,identification and susceptibility test.Results Among 1 745 specimens,896 cases of mycoplasma in-fection were detected with the total detection rate of 51 .35%,including Ureaplasma urealyticum (Uu)infection in 841 cases (48.19% ),Uu combined Mycoplasma hominis(Mh)infection in 92 cases(5.27% )and Mh infection in 17 cases(0.97%).The de-tection rate was 9.46% in males and 41 .89% in females,the infection rate in females was obviously higher than that of males.The results of drug susceptibility test showed that the sensitive rate to doxycycline,josamycin and minocycline was higher than 95%, while the resistant rate to norfloxacin and lincomycin was higher than 75%.Conclusion Mycoplasma infection in this hospital is dominated by Uu.Doxycycline,josamycin and minocycline can be used as the empirical medication to treat mycoplasma infections in this hospital.But before clinical treatment,the mycoplasma culture and the drug susceptibility test should be performed as possible for rational and standardized medication.
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Objective To improve the serum quality controlling level ,in order to regularize and standardize the laboratory processes .Methods Gallery of hemolysis ,lipidemia or icterus serum specimens was established by Roche cobas p612 QSI camera system .The trouble serum specimens were screened out through taking photos of interfere serum and comparing with the gallery . The serum indexes of hemolysis ,lipidemia and icterus were detected respectively as reference information of inspection report .Com‐paring the results of 16 710 specimens detected by system screening and visual judgment ,the accuracy of QSI camera system was verified .Results 195 trouble serum pictures were elected in the gallery .The compliance rates of hemolysis ,lipidemia and icterus se‐rum specimen between QSI camera system and visual judgment were 94 .2% ,87 .4% ,and 60 .9% respectively .Conclusion The QSI camera system can replace visual judgment to screen the trouble serum specimens .
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Objective:To investigate involvement of the Ras/Erk signal pathway in activation of human ??T cells stimulated by Mycobacterium tuberculosis low molecular peptide antigen(Mtb-Ag).Methods:PBMC were isolated from peripheral blood of healthy subjects and simulated in vitro with Mtb-Ag,CD3mAb or PMA and inomysin(IM).CD69 expression of total T cells and ??T cells were measured by flow cytometry at different hours after stimulation, and the number of expanded ??T cells were counted after 10 days of culture. PD98059,a specific inhibitor for Erk pathway was used to treat PBMC for 30 min before stimulation.Results:The proportions of CD69 expressing cells in total T cells and ??T cells were same as 99% at 6 h after stimulation of PBMC with PMA +IM and similar about 55% at 24 h after stimulation with CD3 mAb,whereas those at 24 h stimulation of PBMC with Mtb-Ag were 75.2% and 16.0%,respectively.CD69 expression and proliferation of ??T cells activated by Mtb-Ag were markedly inhibited by pretreatment of PBMC with PD98059.Conclusion:Mtb-Ag is a specific stimulator for activation of ??T cells, and Ras/Erk signal transduction pathway involves in the activation of ??T cells.
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Objective: To investigate the mechanisms of anti-tumor cytotoxicity of human ??T cells activated by Mycobacterium tuberculosis antigen (Mtb-Ag) . Methods: The peripheral blood mononuclear cells (PBMC) of healthy donors were stimulated by Mtb-Ag in vitro to activate preferentially ??T cells that were expanded in IL-2 contained medium.The high purified ??T cells were isolated by the positive selection on MACS separator. The expression of mRNA of perforin, granzyme B and Fas Ligand in purified ??T cells were measured by RT-PCR technique. Fas Ligand on ??T cells and Fas molecule on tumor cells were detected by flow cytometry. The MTT assay was used to measureanti-tumor cytotoxicity of ??T cells. The inhibitory effects of concanamycin A (CMA) for perforin/granzyme pathway, and brefeldin A (BFA) for Fas/Fas ligand pathway on cytotoxicity of ??T cells were observed. Results: The mRNA of perforin and granzyme B and Fas ligand were highly expressed in purified ??T cells. The cytotoxicity of ??T cells against K562 and PG that expressed low level of Fas molecule (6.5% and 7.8%) was remarkably inhibited by pretreatment of the ??T cells with CMA (200 nmol/L) by 72% and 76%, respectively, while the BFA(30 ?mol/L) showed less inhibitory effects by 32% and 25%, respectively; However, it was showed that the BFA (30 ?mol/L) preferentially inhibited the cytotoxicity of ??T cells against tumor cell lines Raji and A375 that expressed high level of Fas molecule on cell surface (98.5% and 70.6%, respectively).Conclusion: Preferentially, granule exocytosis was a main mechanism by which the Mtb-Ag activated ??T cells showed cytotoxicity aganist tumor cells that expressed low level of Fas molecule, whereas both Fas/Fas ligand pathway and granule exocytosis were involved in the cytotoxicicty of Mtb-Ag activated ??T cells against the tumor cells that expressed high level of Fas molecule.